RESUMEN
A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Receptor de la Señal 2 de Direccionamiento al Peroxisoma/metabolismo , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/metabolismo , Peroxisomas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Sistemas Especialistas , Células HeLa , Humanos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptor de la Señal 2 de Direccionamiento al Peroxisoma/química , Receptor de la Señal 2 de Direccionamiento al Peroxisoma/genética , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/química , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/genética , Peroxisomas/enzimología , Dominios y Motivos de Interacción de Proteínas , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteolisis , Proteómica/métodos , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Pex7p is the cytosolic receptor for peroxisomal matrix proteins harbouring PTS2 (peroxisome-targeting signal type-2). Mutations in the PEX7 gene cause RCDP (rhizomelic chondrodysplasia punctata) type 1, a distinct PTS2-import-defective phenotype of peroxisome biogenesis disorders. The mechanisms by which the protein level and quality of Pex7p are controlled remain largely unknown. In the present study we show that dysfunctional Pex7p, including mutants from RCDP patients, is degraded by a ubiquitin-dependent proteasomal pathway involving the CRL4A (Cullin4A-RING ubiquitin ligase) complex. Furthermore, we demonstrate that the degradation of dysfunctional Pex7p is essential for maintaining normal PTS2 import, thereby suggesting that CRL4A functions as an E3 ligase in the quality control of Pex7p. Our results define a mechanism underlying Pex7p homoeostasis and highlight its importance for regulating PTS2 import. These findings may lead to a new approach to Pex7p-based therapies for the treatment of peroxisome biogenesis disorders such as RCDP.